PROJECT SUMMARY How microRNAs regulate the newly described iNKT cell subsets as well as iNKT cell cytokine production and activation are almost completely unknown. This gap in our understanding represents an important challenge, especially when considering the immunological impact of these cells in response to infectious diseases. The long-term goal is to elucidate the physiological significance of iNKT cell subsets in response to infection, as well as long-term chronic inflammation. The overall objective of this specific proposal is to define the role of microRNAs (miRNAs) in iNKT cell subset differentiation and activation. The central hypothesis is that distinct miRNAs regulate differentiation of specific iNKT cell subsets. Moreover, we postulate that certain miRNAs regulate iNKT activation, cytokine production and their anti-inflammatory responses. The rationale for the proposed work is that by understanding how miRNAs regulate iNKT cell subset differentiation, activation and cytokine production, we can begin to understand the relevance of each of these iNKT cell subsets in response to infection and chronic inflammation. Given our strong preliminary data, we will test our hypotheses with the following specific aims: 1) Identify novel miRNAs upregulated during iNKT cell subset differentiation and 2) Determine the role of specific miRNAs in regulation of differentiation, cytokine production and activation of iNKT cells in vivo. Under the first aim, the miRNAs up- and down-regulated as iNKT cells differentiate into their specific subsets will be assessed. Under the second aim, an iNKT cell-specific conditional approach as well as germline-deficient approach will be taken to test the role of miR-155, miR-21 and miR-146a in subset differentiation and activation of these cells. This approach is innovative because it uses novel mouse strains and gene expression analysis to understand how miRNAs shape iNKT cell lineage differentiation and activation. New research possibilities regarding miRNAs and the contribution of iNKT cell subsets to the immune response will be attainable as a result. The proposed research is significant because it will substantially increase our understanding of how iNKT cell subset differentiation is regulated and the role played by distinct miRNAs as this process occurs. By understanding how miRNAs regulate this process, we will have the potential to modulate iNKT cell subsets for better disease outcome. Identification and analysis of the role of miRNAs in NKT cell subset differentiation, activation and cytokine production, will inform our use of iNKT cells in the treatment of human diseases.